Feature Articles

Peering into Subcellular Domains

Super-resolution microscopy is revolutionizing the ability to probe subcellular features as small as single molecules. Surpassing traditional limitations of conventional optical microscopes, these new technologies permit very precise visualization and measurement of features that are below the limits of diffraction.

The super-resolution toolbox includes localization microscopy that identifies the location of individual fluorophores a few at a time, structured illumination microscopy that allows a three-dimensional view, and hyperspectral confocal imaging, among others. Applications for the new technologies range from depicting the fine architecture of cellular immune processes to creation of improved biofuels.

Pavel Tolar, Ph.D., program leader in the division of immune cell biology at MRC National Institute for Medical Research, provides a perspective on several current imaging technologies.

“Traditional fluorescence microscopy is hampered by the resolution limit set by the diffraction of light that is ~200–300 nanometers. Recently, a number of novel instrument-based fluorescence approaches have been employed to circumvent these limitations. We are now seeing imaging down to a resolution that approaches molecular scale (10–15 nm).”

An example of such nanomolar-scale imaging is that of fluorescent resonance energy transfer (FRET), a single molecule technology in which energy is transferred from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor).

“FRET is well-suited to detect protein interactions as well as conformational changes. This provides a remarkable visualization of dynamic protein function to monitor protein-protein interactions (e.g., how signaling complexes assemble) where the distance of interaction is from 2–10 nm. For that reason it is useful for drug screening. But, it is also limited by its low throughput.”

According to Dr. Tolar, the most accessible of these new technologies are photo-activated localization microscopy (PALM) and the related stochastic optical reconstruction microscopy (STORM).

“PALM/STORM are based on the detection and very precise localization of individual molecules. Both employ photo-activatable fluorescent labels for localizing many molecules in a sequential manner using repetitive cycles of activation and imaging. The end result is generation of a high-resolution image that maps the positions of all the molecules monitored. The technology currently is only limited by the number of molecules that can be activated at one time, their brightness, and the rate of photobleaching.”

Deciphering Spatial Relationships

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PALM image of an activated Jurkat T cell: Two signaling proteins are shown. Linker of activation in T cells (LAT) conjugated to the photoactivatable protein dronpa is shown in green, while SH2 domain-containing leukocyte protein (SLP) conjugated to the photoactivatable protein PAmcherry is shown in red. The dots show individual molecules and the insert shows a close-up of the middle portion of the cell. [NCI]

Although signaling complexes have been extensively studied along a number of fronts with numerous biochemical and imaging techniques, many questions remain. An example is signaling downstream of the T-cell antigen receptor (TCR).

Dr. Barr and Eilon Sherman, Ph.D., in collaboration with other colleagues in the LCMB, utilized PALM technology to characterize the organization of signaling molecules during T-cell activation. According to Dr. Barr, “To decipher receptor-regulated cellular signaling (a process involving transient, heterogeneous complexes of undefined structure), we decided to use single and two-color PALM. This is useful particularly to study complexes in the plasma membrane of intact T cells that are downstream of the TCR.”

They first examined the organization of the TCR adapter protein LAT by conventional fluorescence microscopy (diffraction, confocal, and total internal reflection microscopy). Dr. Sherman says they found a few surprises.

“We found that LAT microclusters accounted only for a small portion of the total LAT molecules on the cell. We next employed PALM imaging because it allowed us to observe individual LAT molecules that were tagged with photo-activatable fluorescent proteins. We measured the probability density of locating individual LAT-tagged molecules with a precision of ~20 nm. We also analyzed the scale of LAT clustering using pair-correlation functions. We determined that the majority of LAT molecules were localized to very small nanoclusters of 2–4 detected molecules.”

Dr. Sherman notes that PALM also permitted following of the interactions of the phosphorylating enzyme ZAP-70 and other associated TCR-signaling molecules. Dr. Sherman concludes, “Zap-70 preferentially phosphorylates LAT especially in sites where LAT co-resides with the TCR zeta chain. Overall, our observations provide an example of how PALM technology can help to better discern the complexities of the organization of such signaling complexes as well as the mechanisms involved in shaping how they are arranged in the plasma membrane.”

Blink Technology

A super-resolution imaging technique, Blink Microscopy (Blink), uses sequential, sparsely distributed single molecule imaging to create maps identifying the precise locations of individual molecules down to a level of ~30 nm. “Blink can exploit the fluctuating emission of fluorophores that are able to photoswitch from a bright to a dark state,” explains Aaron K. Neumann, Ph.D., professor, department of pathology, University of New Mexico School of Medicine.

“My colleagues, Ken Jacobson, Ph.D., principal investigator at the University of North Carolina at Chapel Hill, and Philip Tinnefeld, Ph.D., principal investigator at Technische Universitat Braunschweig, and I used Blink to investigate the lateral distribution of a dendritic cell membrane protein called DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin, also called CD209). This protein binds to a large range of pathogens such as HIV and Ebola virus. DC-SIGN presents as tetramers in microdomains on the cell surface.”

Because Blink is based on the sequential localizations of single dyes used for immunofluorescent labeling, the precise location of individual DC-SIGN probes could be determined. “Basically we generate movies taken while the dyes were induced to blink in the presence of reducing and oxidizing agents. Most dye is driven into a dark state under chemical conditions used, while a small number of total dyes activate at one time. Each point in a Blink image represents a precise location of a single molecule generated by fitting the spatial distribution of the signal to a Gaussian function.”

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